Prevalence of α-thalassaemia genotypes in pregnant women in northern Thailand.

BACKGROUND & OBJECTIVES: Alpha-thalassaemias are genetic disorders with high prevalence in northern Thailand. However, common genotypes and current data on the prevalence of α-thalassaemias have not been reported in this region. Therefore, the objective of the present study was to determine the prevalence of α-thalassaemia genotypes in pregnant women in northern Thailand. METHODS: Genomic DNA was extracted from blood samples of pregnant women who came to Maharaj Nakorn Chiang Mai University Hospital during July 2009 to 2010. The common deletion and point mutation genotypes of α-thalassaemia were evaluated by gap- polymerase chain reaction (PCR) and PCR with restriction fragment length polymorphism (RFLP). RESULTS: Genotypes of 638 pregnant women were: 409 samples (64.11%) being normal subjects (αα/αα) and 229 samples (35.89%) with α-thalassaemias. these 229 samples could be classified into deletional HbH disease (--SEA/-α3.7) for 18 samples (2.82%); heterozygous α0-thalassaemia --SEA type (--SEA/αα)) for 78 (12.23%); heterozygous α+-thalassaemia - α3.7 type (-α3.7/αα) for 99 (15.52%); homozygous α+-thalassaemia - α3.7 type (-α3.7/- α3.7) for five (0.78%); heterozygous α+-thalassaemia - α4.2 type (-α4.2/αα) for two (0.31%); and heterozygous HbCS (αCSα/αα) for 27 (4.23%) cases. INTERPRETATION & CONCLUSIONS: The prevalence of α-thalassaemias in pregnant women in northern Thailand was high. This finding supports the implementation of the prevention and control of this common genetic disorder by screening for α-thalassaemia genotypes.

Frequencies of IL10 SNP genotypes by multiplex PCR-SSP and their association with viral load and CD4 counts in HIV-1-infected Thais.

BACKGROUND: Interleukin (IL)-10 is an immunoregulatory cytokine, levels of which can be influenced by single nucleotide polymorphisms (SNPs) in the promoter. Some, but not all previous studies have shown associations of IL10 SNPs with HIV-1 disease progression, using markers such as viral load or CD4 count. There are few data on IL10 SNP frequencies and HIV-1 disease in regions where non-B HIV-1 subtypes predominate. OBJECTIVE: To determine genotypes, haplotypes, allele frequencies and associations with markers of HIV-1 disease progression of ILO SNPs. METHODS: A new multiplexed PCR-SSP assay to detect IL10 SNPs at positions -1082, -819 and -592 was developed and used to determine genotypes and haplotypes in 244 HIV-1 CRF01_AE-infected northern Thais having a median time since HIV-1 infection of 2.7 years. RESULTS: At position -1082 of IL10, AA genotype and A allele were the most common (87.3% and 93.2%, respectively). The -819 CT and -592 CA genotypes were the most prevalent (44.3%), and -819T and -592A were the most prevalent alleles (64.8%). The ATA/ATA was the most common genotype (42.6%) with the most prevalent haplotype of ATA (64.7%). No associations of any of the three ILO SNPs with CD4+ or CD8+ T cell counts or with viral load were found. CONCLUSIONS: This first report of IL10-1082A, -819T and the IL10-592A allele frequencies in HIV-l-infected Thais shows the highest frequencies in HIV-l-infected persons worldwide. The lack of association of IL10 SNPs with CD4+ T cell count and viral load suggest that other genes may influence these markers in HIV-l-infected Thais.

Intracellular growth and drug susceptibility of Mycobacterium tuberculosis in macrophages.

Intracellular growth of Mycobacterium tuberculosis in human and rabbit monocytes and in mouse and guinea pig macrophages was evaluated. Monocytes or macrophages were infected with M. tuberculosis H37Rv with the multiplicity of infection at 10 mycobacteria per monocyte. The average percentages of infected human and rabbit monocytes were 22% and 19%, while mouse and guinea pig macrophages were 46% and 58%, respectively. The average generation times of M. tuberculosis H37Rv inside human and rabbit monocytes and in mouse and guinea pig macrophages, after culturing the infected cells for 10 days, were 33.4, 50.3, 31.4, and 25.6 h, respectively. Using infected guinea pig macrophages for intracellular evaluation of drug susceptibility, the minimal inhibitory concentrations (MICs) of isoniazid to the intracellular and extracellular M. tuberculosis H37Rv were 0.1 and 0.4 microg/ml, while the MICs of rifampicin were 0.1 and 0.2 microg/ml, respectively. The minimal bactericidal concentrations (MBCs) of isoniazid to the intracellular and extracellular H37Rv were 0.2 and 0.4 microg/ml, while the MCSs of rifampicin were 0.1 and 0.2 microg/ml, respectively.

Production of a mouse hybridoma secreting monoclonal antibody highly specific to hemoglobin Bart's (gamma4).

Hemoglobin (Hb) Bart's (gamma4) was isolated and purified from Hb Bart's hydrops fetalis syndrome blood by CM-Sephadex C-50 chromatography. The isolated Hb Bart's was analyzed for its purity by high performance liquid chromatography. Balb/c mice were immunized intraperitoneally with Hb Bart's. The immunized mouse splenic cells were hybridized with mouse myeloma, X63-Ag8.653, by polyethylene glycol. There were 12 hybridoma clones, out of several thousand culture wells, secreting antibody against purified Hb Bart's. However, when those 12 monoclonal antibodies (mAb) were tested with Hb Bart's (gamma4), HbF (alpha2gamma12), HbH, HbE, and HbA2, there was only 1 hybridoma clone secreting mAb highly reactive to Hb Bart's with very low reactivity to HbF. A rabbit polyclonal antibody with relative high reactivity to Hb Bart's compared to HbF with the ratio of 2.4:1 was also produced by affinity column chromatography for the purpose of developing an enzyme-linked immunosorbent assay (ELISA) base for qualitative and quantitative determination of Hb Bart's in adult hemolysates. Preliminary results in quantitative determination of Hb Bart's in Hb solution of 3 alpha thalassemia families having at least 1 child with HbH disease and 6 normal subjects indicated that it was possible to quantify Hb Bart's by our developed ELISA with appropriate sensitivity and specificity.

Lymphocyte transformation in lepromatous leprosy: a study of the influence of disease activity and symptom duration

The lymphocyte hyporesponsiveness to M. leprae of patients with active lepromatous leprosy has been well described. This immune defect is less well understood in terms of its time of origin, possible reversibility and specificity. To further examine the persistence and specificity of this abnormality, lymphocyte transformation tests of 93 leprosy patients to lepromin, BCG and PHA were studied. Among lepromatous patients, a decreased response to M. leprae was seen, whether the disease was active or inactive. Decreased responses to BCG were found in lepromatous patients with active disease, but not in those with inactive disease. The duration of patient symptoms was not associated with differences in LTT responses among the active lepromatous patients.